During the past year, we we have completed studies with Dr. Katy Rezvani using ELISPOT techniques to quantitate the incidence of T-cells with specificity for each of 4 HLA-A2-restricted epitopes of the tumor related peptide PRAME in normals and patients who received an allotransplant for treatment of acute or chronic leukemia. These studies documented the presence of PRAME-specific T cells in patients with leukemia and suggested PRAME epitopes might be attractive targets for T cell adoptive transfer and anti-tumor vaccine strategies. Analogous studies using Elispot methods were begun in collaboration with Agnes Yong of Dr. Barrett's laboratory studying the immunogenic potential of HLA-A2 binding peptides derived from the polycomb group proteins BMI-1 and EZH2. These molecules which play a role in regulating proliferation, and hence are abundantly expressed in immature CML cells. T cell Elispot responses to BMI-1, and to a lesser extent EZH2, could be detected in normal individuals, and in donor-derived cells from the blood of patients with chronic myelogenous leukemia (CML) after allotransplantation. Unlike other potential immune targets such as the myeloid granule proteins proteinase 3 and elastase which can be down-regulated in tumor cells without adversely affecting cell viability, polycomb proteins are essential for CML proliferation. Consequently tumor cells cannot simply evade immune destruction by polycomb specific T cells through down regulation of expression. Additional studies investigating the feasibility of using these molecules as targets for leukemia immunotherapy are warranted. During the past year we've systematically compared the impact of two clinical grade reagents, CD3/CD28 coated magnetic beads and OKT3 anti-CD3 antibody as reagents for expanding human T cells ex vivo. CD3/CD28 beads have been used extensively because T cells are known to respond vigorously to simultaneous stimulation of a costimulatory molecule (CD28) plus the T cell receptor. By contrast, isolated stimulation of the T cell receptor complex without costimulatory signaling can promote T cell apoptosis or anergy. Although OKT3 alone cannot stimulate costimulatory signals, we found that peripheral blood T cells exposed OKT3 in the presence of additional antigen presenting cells (APCs) proliferated at least as well as CD3/CD28 bead-stimulated cells. Furthermore, in studying the phenotype of the expanded cells using flow cytometry, OKT3 stimulated CD8 cells demonstrated greater diversity and expressed higher levels of CCR7, CD27, and CD45RA than cells stimulated with CD3/CD28 beads suggesting greater preservation of nave and central memory cells. Significantly, in comparing the efficacy of CD3/CD28 beads and OKT3 plus irradiated autologous APCs in re-stimulating T cells previously stimulated ex vivo, OKT3 proved to be markedly more effective. We hypothesize that the greater efficacy of OKT3 plus irradiated autologous APCs reflects the ability of OKT3 stimulated T cells to also interact with a diverse set of costimulatory molecules and cytokines expressed by APC. Based on our studies we suggest the OKT3/APCs combination may have advantages, at least by comparison to CD3/CD28 magnetic beads, in ex vivo expansion of T cells for use in immune reconstitution and/or adoptive immunotherapy.